Method of producing ribonucleic acid



United States Patent 1 Claim. (01. 19s-2s This invention relates to methods of producing ribonucleic acid.

The international chemical abbreviation for ribonucleic acid is -RNA and the same will sometimes hereinafter be so abbreviated.

Unless otherwise stated, increments of measure, used in this specification, such as units of percentage or parts, are units by weight.

It has been known for some time that a main part of yeast nucleic acid is RNA. Recently it has been discovered that RNA itself, apart from its presence in yeast, is useful in seasoning agents, nutritive foods, medicines, etc. RNA has furthermore recently occupied an important position biochemically, and numerous reports have been made concerning its biosynthetic mechanism.

Yeast is and has been regarded as the principal source of supply of RNA. However, previously, RNA has been viewed as merely a constituent element of yeast, and no one has paid any particular attention to increasing the RNA yield of a given quantity of yeast. The primary object of this invention is to obtain RNA on a oommerical scale by carrying out yeast culture in such a manner as to render the production of RNA commercially feasible.

Various kinds of yeasts, for bakery items, foodstuffs, etc., have been commercially produced, and the principal techniques for the production of such yeasts on a commercial basis is well understood. '1 was not satisfied with the yield of RNA which had been derived from previous yeast cultures, and have experimented and investigated efiects of the addition of various kinds of inorganic and organic compounds to saccharine media in an effort to increase the yield of RNA. I discovered that the addition of zinc ion to the culture liquid greatly increased the rate of RNA formation and the yield of RNA relative to the volume of supplied and consumed sugar without having any appreciable adverse effect on the yeast growth rate or yield. -It is therefore a further object of my invention to facilitate the production of RNA by the addition of zinc ion to the culture liquid.

I recognize that others have previously noted various relations between the zinc ion and the growth metabolisms of micro-organisms. According to these results obtained by others, zinc ion has an important stimulating effect on the growth of yeast; is an essential composition as a co-enzyme for various kinds of dehydrogenases', and likewise has the eifcet of increasing the rate of sugar consumption. However the effects of zinc ion on the formation of RNA up to the time of my experiments and investigations, were quite unknown. I discovered that by combining a concentration of zinc ion and that of phosphoric acid, with proper coefficient of oxygen absorption rate, the formation of RNA would be markedly increased.

The coefiicient of oygen absorption rate is internationally chemically abbreviated as Kd--. The term Kd as used in this test means the result to be obtained by the method set forth in the report by Yamada et al. in volume 27, pages 704-708, of the 1956 reports of the Japanese Agricultural Chemical Society.

3,272,714 Patented Sept. 13, 1966 It has been previously recognized that phosphoric acid has a positive effect in promoting the rate of RNA yield. Such yields of RNA which have been previously obtained, by use of phosphoric acid, have, however, not been sufficient to render the same commercially feasible. 1 discovered that by suitably combining the zinc ion concentration with a phosphoric acid concentration, the yield of RNA cauld be promoted to such an extent as to provide a commercially practicable process for the production of RNA.

It has likewise previously been recognized that a high Kd value results in an increased yield rate of yeast and growth rate of yeast. I discovered that the addition of a zinc ion, when the Kd value is high, manifestly increases the yield of RNA.

The general results of my experimentations and investigations is that when the three factors--zinc ion concentration, phosphoric acid concentration, and Kd are combined, the yield rate and the formation rate of RNA will be promoted to the extent whereby RNA may be profitably produced on an industrial basis.

I recognize that there are many substances which have been effectively utilized for accelerating the growth rate of yeast, such as various kinds of vitamins, amino acids, nitrogen sources, etc., however, these substances do not produce an effect which will elevate the rate of RNA yield in relation to sugar. Prior knowledge in the culture of yeast has therefore failed to produce any process whereby the yield rate of RNA relative to sugar and the rate of RNA formation could be practicably carried out on an industrial scale.

Optimum results in the production of RNA are obtained when zinc ion concentration in the culture liquid is over 0.25 ppm; phosphoric acid is added to the culture liquor in the amount of 0.15%, by weight, or above; and Kd is over l.0 l0

For example, well known kinds and concentrations of sugar; nitrogen sources; potassium salts; magnesium salts; inorganic substances, other than zinc; culture temperatures; pH; Kd; and combinations thereof, or various kinds of vitamins, amino-acids, and other additives for growth acceleration, etc., as well as known culture conditions which will have effects of growth acceleration, are compatible with my process and may be utilized under appropriate conditions.

Since RNA is normally accumulated in the body of yeast, it is generally the practice to indicate the progress of yeast culture by the content of RNA, the amount of yeast formed, the growth rate, the yield rate, etc., relative to supplied sugar or consumed sugar. However, in this specification, in the case of culture of batch systems, the yield rate of RNA is judged by measuring and recording the RNA yield rate in relation to the consumed sugar, and the rate of RNA formed by the weight of RNA formed per unit volume of the liquor on the basis of time elapsed, respectively. As it is known that the formation of RNA is richest in the yeast growth period, the amount of yeast and the amount of residual sugar are also mentioned so as to facilitate the understanding of the growth curves.

The following description deals with the effects of zinc ion in sequence. As to zinc to be added, since it is usual that the pH of the culture is on the acid side, regardless of whether or not the zinc to be added is in a non-ionic condition, the desired results will be obtained with any composition of zinc, so long as such zinc composition is of a nature which can be ionized in the culture liquor.

In the culture results reported below, Candida utilis was used as the control yeast, and its yeast mash was added to the present culture liquor for 3%, then shakeculture or aeration-agitation culture was carried out on the batch system under regulated conditions so that the zinc ion concentration in the culture liquor would be 0.05 p.p.m., and also under an aerobic condition where the Kd value was 12.6 x This provided a control medium by which results of my experiments could be measured. 10

with no addition of inorganic salts and the media with addition of inorganic salts in respect of the rate of sugar 4 consumption of yeast, and the rate of formation of RNA and the yield of RNA.

For example, in the case of a culture for 11 hours, an effect as :high as 200% has been indicated in the yield rate of RNA, and an effect as high as 300% in the rate of formation. The efiiects of addition of the vitamins and others in the media (with addition of inorganic salts may be noted to some extent, but this is relatively inconsequential.

Table 2 tabulates the experimental results attained when the 3 kinds of inorganic salts mentioned in Table 1 were added to synthetic media, individually or in combinations. Iron and copper ions have shown almost no effect, whereas the effect of the zinc ion was distinctive. For example, in a medium after a culture of 1 0 hours, the yield rate of RNA and the rate of RNA formed was indicated to be as high as 200% or more in comparison with the control.

As seen in the table, effects of zinc ion addition are elevated by mixing with copper ion or copper and iron lons.

TABLE 1 Time elapsed 9 Hours 11 Hours Result Residual RNA RNA Amount RNA Residual RNA RNA Amount RNA Sugar Amount Yield of Yeast Content; Sugar Amount Yield of Yeast Content (percent) (mg/lit.) (percent) (percent) (percent) (percent) (mg/lit.) (percent) (percent) (percent) Medium N 0.:

NOTE.C0mposition of Respective Culture Medium Medium No.:

1. Fundamental medium: (Glucose 3.0%, (NHmHPOi 1.0%,

KH POi 0.00%, MgSOflHgO 0.05%). 2. Fundamentaljmedium plus Sodium citrate 0.1%. 3. Fundamental medium plus Asparagiue 0.25%. 4. Fundamental medium plus Vitamins of various kinds (Biotine 5. Fundamental medium plus Vitamins of various kinds plus Inositol mg./lit.

6. Fundamental medium plus Inorganic salts of various kinds (ZnSOflH O 6 mg./lit., F0(NH4)z.(SO4)z.6H2O 3 mg./lit., CHSOljHzO 0.3 mg./lit.).

7. Fundamental medium plus Inorganic salts of various kinds plus Vitamins of various kinds plus Inositol 30 mg/lit.

0.06 mg./lit., Calcium Pantothenate 1.5 mg./lit., Yitamiu 8. Medium (7) plus Asparagine 0.25% B hydro-chloride 13.2 mg./l1t., Pyridoxine 3.6 mg./l1t.). 9. Medium (7) plus Asparagine 0.25% plus Sodium citrate 0.1%.

TABLE 2 Time elapsed 8 Hours 10 Hours Result Residual RNA RNA Amount RNA Residual RNA RNA Amount RNA Sugar Amount Yield of Yeast Content Sugar Amount Yield of Yeast Content (percent) (mg/ht.) (percent) (percent) (percent) (percent) (mg/lit.) (percent) (percent) (percent) Medium No.:

Nora-Composition of Respective Media Medium N 0.:

1. Fundamental Medium (Glucose 3.0%, Urea 0.30%, Ammomum Sulphate 0.10%, KH2PO4 0.15%, MgSO47HzO 0.05%, CaClgZHzO 0.02%).

2.0F7undame nta1 Medium plus ZnSO -7H1O 3.0 mgJlit. (As Zn ppm.

3. Fundamental Medium plus ZnSO47H O 12.0 mgJlit. (As

Zn 2.7 p.p.m.).

4. Fundamental Medium plus ZnSOflHzO 48.0 mg./lit. (As

Zn 10.9 p.p.m.)

5. Fundamental Medium plus Fc(NH4);(SO -6H2O 2.0.

6. Fundamental Medium plus Fe(NH4) (SO4) -6H2O 8.0.

7. Fundamental Medium plus Fe(Nl-I4)z(SO4)z-6II O 32.0.

8. Fundamental Medium plus CuSO -5H1O 0.3.

9. Fundamental Medium plus CuSOll-5H O 1.2.

10. Fundamental Medium plus 01180451110 4.8.

11. Fundamental Medium plus ZnSO -7H2O 6.0 mgjlit. plus Fe(NH4)2(SO4) 2-6HZO 4.0 mg./lit.

12. Fundamental Medium plus ZI1S04-7II2O 6.0 mgJlit. plus CJSO4-5H2O 0.30 mgJlit.

13. Fundamental Medium plus F0(NII4)z(SO4)g-6Hz0 4.0 mg./

llt. plus CHSOA5HQO 0.30 mgJlit.

14. Fundamental Medium plus ZnSO4-7H20 6.0 mgJlit. plus,

Fe(NH4)Z'6II2O 4.0 mg./lit. plus CuSO4-5II O 0.30 mgJlit.

ion. My experiments and investigations lead me to believe that vitrually any reasonable concentration of zinc ion will accomplish the purposes of my invention, and I have therefore specified the zinc ion only at the lower 5 limit, 0.25 p.p.m.

TABLE 3 Time elapsed 8 Hours Hours Result Residual RNA RNA Amount RNA Residual RNA RNA Amount RNA Sugar Amount Yield of Yeast Content Sugar Amount Yield of Yeast Content (percent) (mg/lit.) (percent) (percent) (percent) (percent) (mg/lit.) (percent) (percent) (percent) Medium No;

N 0TE.C0mposition of Respective Media: 1.0 p.p.m.).

Medium No 8. Fundamental Medium plus ZnSO4.7H 8.8 mg./lit. (as Zn 1. Fundamental Medium (Glucose 3.0%, Urea 0.30%, Ammonium Sulfate 0.10%, KH2PO4 0.15%, MgSO4.7H:O 0.05%, CaClz.2HzO 0.02%

2. Fundamental Medium plus ZnSOMH O 0.55 mg./lit. (as Zn 0.125 p.p.m.).

3. Fun amental Medium plus ZnSOMH O 1.10 nag/lit. (as Zn 0.25 p.p.m.).

4. Fundamental Medium plus ZnSOaJHzO 1.65 mg.[lit. (as Zn 9.37 p.p.m.).

5. Flndamental Medium plus ZnSOMHZO 2.2 mg./1it. (as Zn .5 p.p.m. 6. Fundamental Medium plus ZHSO4.7HzO 3.3 mg./lit. (as Zn .75 p.p.m.

7. Fundamental Medium plus Z11S04.7H2O 4.4 mgJlit. (as Zn tion shows marked effects on the rate of formation in the yield of RNA as compared with the control medium containing only 0.05 p.p.m. of zinc ion.

As regards the upper limit of zinc ion concentration,

I have determined no specific limitation. As shown in the table, in {which a medium was utilized having a concentration of zinc ion in excess of 1,000 p.p.m., the same did not exhibit any particular obstructive effect. Zinc ion is therefore considered to remain effective over a broad range of concentrations higher than 0.25 p.p.m. As to the optimum eflective concentration, I experimented with a broad range of concentrations, but was unable to reach a conclusion as to the optimum concentration of zinc 2.0 p.p.m. 9. Fundamental Medium plus 211501711 0 17.6 mg./lit. (as Zn 4.0 p.p.m. 10. Fundamental Medium plus Z11S04.7Hz0 70.4 mg./lit. (as Zn 16 p.p.m.).

11. Fundamental Medium plus ZHSOJJIIQO 282 mg./lit. (as Zn 64 p.p.m.

12. Fundamental Medium plus ZnSO .7H;0 1,128 mg./lit. (as Zn 256 p.p.m.).

13. Fundamental Medium plus ZnS04.7H O 2,256 mgflit. (as

Zn 512 p.p.m.).

14. Fundamental Medium plus ZDSO4.7H2O 4,512 mg./lit. (as

Zn 1,024 p.p.m.).

Actual results of my experiments prove the fact that the yield rate of RNA is increased by the addition of zinc ion. Present knowledge about the effects of zinc ion on micro-organisms, particularly yeast, is limited to the extent that it is impossible to explain the abnormal high effects of zinc ion addition on the formation of RNA, but factual results prove this to be true. One of the achievements of my invention is the discovery of the existence of the close relationship between the zinc ion and the mechanism of biosynthesis of RNA.

I have further determined that the efiects of the addition of zinc ion are not limited to any specific genus or species of yeast, but that such effects are common among TABLE 4 Time Elapsed. 11 Hours 15 Hours Strain Used Result Residual RNA Yield of Amount RNA Residual RNA Yield of Amount RNA Sugar Amount RNA of Yeast Content Sugar Amount RNA of Yeast Content (percent) (mg/lit.) (percent) (percent) (percent) (percent) (mg/lit.) (percent) (percent) (percent) Saccharomyces cercvz'st'ae 0.44 237 0.93 0.44 5. 4 0.10 216 0. 74 0.50 4. 3 0. 10 696 2. 4 0. 53 13. 2 0. 10 658 2. 3 0. 56 11. 8

Saccharomyces carlsber 0.74 212 0. 94 0.35 6.1 0.26 52 0.76 0. 40 5. 2 gensis 0. 10 609 2. 1 0- 47 12. 9 0. 10 116 2. 2 0. 56 11. 6

Saccharomyccs chcvalz'eri 0. 34 242 0.91 0. 39 6. 2 0.16 225 0.79 0.46 4. 9 0. 10 580 2. 0 0. 44 13. 2 0. 10 617 2. 1 0. 45 13. 8

Hansenula anomala I 1.08 392 2.0 0.74 5. 3 0.10 271 0.94 0.52 5. 2 II 0. 10 453 1. 6 0. 57 8.0 0. 10 397 1. 4 0.61 6. 5

Hansemtla schnegii. I 2. 25 207 2. 8 0.43 4. 8 1. 80 225 1. 9 0.64 3. 5 II 2. 00 326 3. 3 0. 34 9. 2 0. 21 733 2. 6 0. 92 8. 0

Rhodotorula glutt'm's I 2.90 60 6.0 0.10 5. 9 2. 151 7. 6 0.27 5. 6 II 2. 104 10. 4 0. 08 12. 9 2. 60 289 7. 2 0. 29 9. 9

Candida utilis I 1. 44 451 2. 9 0.55 8. 2 0.10 476 1. 6 1. 33 3. 6 II 0. 10 651 2. 2 0. 68 9. 6 0. 10 590 2. 0 1. 63 3. 6

NoTE.-C0mposition Culture of Media:

1. Glucose 3.0%, Urea 0.3%, Ammonium sulfate 0.1%, KHzPO4 0.15%, MgSO4.7HzO 0.05%, CaCl2.2HzO 0.025%, Fe(NH4)2(SO4)2.6H2O 4 mg./lit.,

L()asparagine 0.25%, Biotine 0.66 mg./lit., Calcium pantothenate 1.5 mg./lit., B1hydrochloride 13.2 mg./lit., Pyridoxine 3.6 mg./lit., Inositol 30 mg./.1t. II. I+ZnSO .7H2O 20 mg/lit.

sporogenous yeasts belonging to the genus Saccharomyces, Zygosaccharomyces, Hansenula, Pichia, Willia, etc., and asporogenus yeasts belonging to the genus Torula, Mycotorula, Torulopsis, etc. Table 4 shows the effects of zinc 8 yeast to be used is not lacking the enzyme inverting cane sugar. As previously set forth, in the case of glucose media, with addition of zinc, the effects of mixing ions of iron, copper, etc., and the mixing of vitamins of vari- 1011 on Yeasts of Various 5 ous kinds, asparagine, sodium citrate, etc., may be noted 5 Fffects of qg 8 Zmc 15 not to some extent. There is likewise some effect achieved by to t i a S i f on 9" ntutntlvi the intermixing of casamino-acid, corn-steep-liquor, soyg sfg s i g i z e ype o bean cake extracts, as indicated in Table 5. The effects e y of these additives is principally related to the increase in In addition to the above mentioned, even though 1on1zed 10 the rate of RNA formed as the result of Shortenin the aluminum, manganese, cobalt, molybdenum, antimony, d f d th th f t d d g h silver, tin, gold mercury, lead, etc., may be added, there perm o m 9 z i 9 yeas re ucmg e is found no positive effect Worth mentioning sugar consumption an cu ture tune, as as promotlng As regards the carbon source, in the case of substances the growth rate of Yeast But these addltlves were fol'md other than glucose, the effect of zinc ion is the same. For almost totally lneflectlvfi for elthef lncfeaslng the yleld example it is true in the case of cane sugar so long as the rate or the RNA content, and thus their effects are believed TABLE 5 Time elapsed 7 Hours 9 Hours Result Residual RNA RNA Amount RNA Residual RNA RNA Amount RNA Sugar Amount Yield of Yeast Content Sugar Amount Yield of Yeast Content (percent) (mg/lit.) (percent) (percent) (percent) (percent) (mg/lit.) (percent) (percent) (percent) Medium N 0.:

Norm-Composition of Respective Media 6. Fundamental medium I plus Casarnino acid 0.20%.

Medium No.2 7. Fundamental medium 11 (Fundamental medium plus 1. Fundamental medium I (Glucose 3.1%, Urea 0.30%, Am- ZnSO4. 71120 0.0 mg./lit.).

monia sulfate 0.10% KHzP04 0.15%, MgSOMH O 0.05%, 8. Fundamental medium II plus Com-steepliquorextract 0.05%. 09.012.21'120 0.02%). 9. Fundamental medium II plus Corn-stcephquorextract 0.20%. 2. Fundamental mediurnIplus Corn-steep-liquorextract 0.05%. 10. Fundamental medium II plus Soy-bean cake extract 0.05%. 3. Fundamental mediumIplus Com-steep-liquor extract 0.20%. 11. Fundamental medium II plus Soy-bean cake extract 0.20%. 4. Fundamental medium I plus Soy-bean cake extract 0.05 12. Fundamental medium II plus Casamino acid 0.20%. 5. Fundamental medium I plus Soy-bean cake extract 0.20 to be substantially different from those of the zinc ion. Table 6 indicates the relationship between the principal However, it should be understood that the effects of their saccharine materials and the addition and non-addition of mixed uses may be utilized in the industrial embodiment zinc ion. In almost genuine saccharine materials such as of the present invention. 45 glucose, cane sugar, etc., the effect of the addition of zinc The measuring of zinc ion concentration in the culture ion is remarkably notable. In saccharine materials conliquor is usually performed by using the polarographic taining impurities of various kinds such as saccharified liqmethod, or by the quantitative colorimetric method, such nor of wood, sulfite pulp waste liquor, molasses, etc., the as the Dithizone method, after the sample has been turned effect of the zinc ion is not so significant as compared with to ashes, then ionizing the ashes. Therefore, it is to be 50 genuine sugar. The zinc concentration obtained by the noted that the difference of ionic or non-ionic character of analytical result of the molasses material differs, dependadded zinc in the cultured broth cannot be detected from ing upon the classification of the molasses. For example, the present analytical method. when starting sugar concentration is 3%, the zinc con- TABLE 6 Time Amount Amount; Yield of Amount RNA Medium ZIISO4.7H2O Elasped of Residof RNA RNA of Yeast Content (p.p.m.) (Hour) ual Sugar (mg/lit.) (percent) (percent) (percent) (percent) Molasses 0 6 0. 29 768 3. 9 1. 07 7. 2 8 0. 22 604 3. 4 1. 22 5. 7

Sultite Pulp Waste Liquor 0 8 1. 90 323 8.1 0. 14 23. 3 10 1. 59 551 7. 9 0. 44 12.7

saccharified Liquor of 0 8 1. 30 703 4.1 0. 83 8. 5 Wood. 10 0.81 818 3. 7 1. 05 7. 8

Strain Used: Candida utilis.

Molasses Medium: Sugar concentration 2.25%, Urea 0.20%, Ammonia sulfate 0.07%, Phosphoric acid 0.3%. Sulfite Pulp Waste Liquor Medium: Sugar concentration 2.30%, Urea 0.3%, Ammonium sulfate 0.07%,

Phosphoric acid 0.3%.

saccharified Liquor of Wood: Sugar concentration 3.0%, Urea 0.3%, Ammonium sulfate 0.07%, Phosphoric acid 0.3%, Corn-steep-liquor 0.15%.

centration is mainly 0.52.0 ppm. The reasons why satisfactory effects of zinc ion addition are found in molasses media containing fairly sufiicient concentration of zinc are not clear. One hypothesis is that zinc in the molasses is present in a difli-cult state of utilization in culture process and the zinc ion concentration in the broth is insuflicient. The other is that added zinc ion inactivates by precipitation of some substance in molasses which inhibits formation of RNA. In either case, it is to be understood that whereas the addition of zinc ion has almost no influence upon the growth rate in various kinds of saccharine material media, the addition of zinc exhibits significant eifect on the production of RNA.

The relationship between the addition and non-addition of zinc ion and the phosphoric acid concentration is shown in Table 7. The efiect of addition of zinc may be seen with molasses of different origins. In order to achieve a high yield rate of RNA, it will be noted that the phosphoric acid concentration is insufficient at 0.10%, and is preferably over 0.15%. RNA may be effectively produced by combining the zinc ion concentration and the phosphoric acid concentration.

The above descriptions dealt with the effects of addition of zinc ion under higher Kd condition in the batch system culture by means of saccharine media of various kinds. However, these efiects are not limited to the batch-system culture, but they may be exhibited entirely in the same manner in other culture systems, such as semi-continuous system (feeding culture, etc.), continuous system, etc.

The essential point in the economy of RNA production by a continuous process may well be expressed by the yield of RNA, and productive efficience of definite facilities. In this invention, the yield rate of RNA (Y can be calculated by multiplying the yield rate of yeast (Y by the content of RNA (R). On the other hand, it is natural that the production efiiciency of RNA (,LLVXR) has a significant relationship with the growth rate of yeast 1), the volume of liquor in the tank (V), the concentration of yeast (X) and the content of RNA (R). Therefore, in the economical production of RNA, it is desirable to increase Y R, ,u, V, and X as much as possible.

TAB LE 7 HaPOi ZHSO4.7H2O Residual RNA Yield Amount RNA Origin of Molasses (percent) (p.p.m.) Sugar Amount of RNA of Yeast Content (percent) (mg./lit.) (percent) (percent) (percent) India 0. 1 18 0. 28 797 4. 1. 07 7. 0. 18 0.27 1, 066 5. 3 1.04 10.3

Indonesia 0. 1 18 0. 22 767 3. 8 1. 04 7. 4 0. 15 18 0. 20 1,070 5. 2 1. 06 10.2

Philippines 0. 1 18 0. 25 815 4. 1 1. 04 7. 8 0. 15 18 0.22 1, 068 5. 3 1. 05 10. 1

The value as when 8 hours have passed.

Fundamental Medium: Starting sugar concentration (Indian molasses 2.28%, Indonesian molasses 2.26%,

Philippine molasses 2.23%). CuSO4.5I-I2O 1.2 mg./lit.

Strain: Candida utilz's.

Table 8 shows the relationship between Kd and zinc ion. It is to be noted that when Kd is below 10 1O' the efiect of zinc ion on production of RNA is relatively insignificant whereas in the case of Kd over 10 10' it is shown that zinc ion has a marked effect on the yield of RNA and the rate of its formation. In higher conditions of Kd, this eflfect incorporates those efiects upon the yield rate of yeast and the rate of its growth. However, it is obvious that the added Zinc directly takes effect as a strong stimulus, as may be seen by the noted increased RNA content.

Urea 0.29%, ammonium sulfate 0.07%, FG(NH4)2 (800261110 4 mg./lit.,

Among these 5 kinds of indexes, the techniques to promote Y ,u., V, and X, are substantially the same as those techniques of yeast culture utilized for foodstuffs or feedstuffs. Hence, in the present invention, explanations will further 'be made in relation to the effects of the invention on the 5 itemsnamely, Y R, Y and VXR. The classification of the yeast, raw materials, and their culture conditions, which can be used in the present continuous culture, are substantially those which may be assumed from the batch culture system, there being no substantial difference.

TABLE 8 Time Residual Content Yield Amount Content Kd Medium Elapsed Sugar of of of of (Hour) (Percent) RNA RNA Yeast RNA (mg/lit.) (percent) (percent) (percent) 13.2X1(H I 6.5 0.56 737 4.4 0.80 9. 2 8. 0 0. 22 885 4. 4 1. 13 7. 9

NOTE:

Strain Used: Candida arborea.

fate 0.10%. (II) I ZnSO4.7H2O 15 mgr/lit.

As indicated in Table 9, in continuous culture, under an aerobic condition with Kd over 1. l0 when the concentration of added zinc ion in the feeding liquor is maintained over 0.25 p.p.m., Y and ,a will show almost no change, but the content of RNA will markedly increase. Along with the known fact that Y and ,u will show good results under a condition of high Kd, significant effects will also be seen with respect to the indexes of Y and ,uVXR.

In Table 10, there are shown examples of continuous cultures by the use of saccharified liquor of wood, sulfite pulp waste liquor, and waste molasses of cane sugar. As shown in this table, the effect of the addition of zinc ion is significant in saccharified liquor of wood and sulfite pulp waste liquor, as compared with molasses. It is believed that the reason why the eflect is diminished in the case of molasses is that the activity of effect of the zinc ion may have been lowered owing to various impurities contained in the molasses raw material.

The following are some examples of the carrying out of my invention, by tank culture. It is to be understood that my invention is not limited by these examples, but they are given only as exemplary methods of the practicing of my invention.

Example 1 culture liquor or Candida arborca was inoculated, and

under the condition of agitation at 150 rpm, and aeration at 100 lit/min. (Kd=25.9 l0 culture was performed. While pH was being adjusted to 4.5 with caustic soda and sulfuric acid when it overstepped the range of TABLE 9 Kd Zn ion Y}; R Y

(p.p.m.) (Percent) (percent) (percent) 3.2 X 10* 0. 125 35. 6 5. 4 1. 9 0. 28 3.2 X 10". 0. 37. 2 6. 0 2. 2 0. 3.2 X 10 0.50 35.2 6. 4 2. 3 0.28 6.2 X 10 0. 125 43. 6 5. 9 2. 6 0. 34 6.2 X 10:"- 0. 25 44.8 5. 5 2. 5 0.32 6.2 X 10 0.50 46. 1 6. 1 2. 8 0.32 10.6 X 10- 0. 125 53. 1 5.1 2. 7 0.34 10.6 X 10'- 0. 25 52. 6 7. 6 4. 0 0. 36 10.6 X 10 0.50 53.0 10.1 5.4 0.33 26.8 X 10' 0.125 56. 1 5. 3 3. 0 0.42 26.8 X 10' 0.25 53.1 7.8 4.1 0.43 26.8 X 10- 0.50 52.8 10.1 5.3 0.43

Strain Used: Candida arborea.

be added as ZnSOMHrO.

Yeast Concentration: Amplitude of Variations 1.82.2%, X of pVXR is to be calculated Volume of Liquor in the Tank: to be calculated as 1 m TABLE 10 Zn ion Y R Y VX R Percent- Medium Added (per- (per- (perp (kgJmfl/Hr.) ages Yn (p.p.m.) cent) cent) cent) (percent) Saccharified Liquor of Wood Control 54. 3 7. 2 3. 9 0.31 0. 335 (100) Sulfitc Pulp Waste Liquor Control. 46. 1 6. 9 3. 2 0. 33 0. 342 (100) Cane Sugar Waste Molasses..." Control. 56. 3 8. 6 4. 8 0.41 0. 529 (100) Strain Used: Candida utz'lz's. Composition of the Supplied Liquor (Sugar Concentration 3-5%) 1. Saccharified Liquor of Wood: Corn-steep-liqnor 0.2%, Urea 0.2%, Potassium phosphate 0.15%, MgSOMHzO Pulp Waste Liquor: Soy-bean cake extract 0.2%, Urea 0.3%, Potassium phosphate 0.20%,

. S MgSOrJHaO 0.05%, CaCl1.2H2O 0.04%.

3. Molasses Liquor of Philippine Origin: Urea 0.2%, Phosphoric acid 0.2%, Ammonium sulfate 0.1%.

Yeast Concentration: 1.3-1.6%, X of VXR is to be calculated as 1.5%. Volume of Liquor in the Tank: To be calculated as 1 m The content and technical meaning of my invention is 7 4.5-5 .0, the residual sugar, concentration of yeast and content of RNA were measured at definite time intervals so as to follow up the progress of culture. The concentration of yeast reached a maximum in 12-13 hours irrespective as to addition of zinc ion. Although almost no difference was noted with the maximum yeast concentration,

13 curve of sugar consumption and growth rate, the RNA content of the section with Zinc ion added indicated 11.0%, whereas that of the section without addition of zinc ion was 7.2%.

To each of the said yeast (corresponding to 1 kg. as dried product), was added 20 lit. of 0.5% NaCl water which was boiled, then extracted at 100 C. for 2 hours. After concentrating the separated supernatant liquor, the extract was subjected to the alcohol treatment. From the former, 69 g. (RNA conent: 79%) of crude RNA was obtained, and from the latter, 102 g. of crude RNA (RNA content: 82%) was yielded.

Example 2 In a stainless aeration-agitation culture tank with a 200 lit. capacity, the effects of addition of zinc ion were examined with molasses of Siamese origin, Philippine origin, and the saccharified liquor of wood from needleleaved tree. In the instance of a starting sugar concentration of 3.0%, the zinc concentration deriving from the said raw materials were calculated to be 1.3 p.p.m., 0.8 ppm. and 0.6 p.-p.m., respectively. The said raw materials were prepared into culture media for 120 lit. each section at a sugar concentration of 3.0% with addition of ammonium sulfate 0.05% and phosphoric acid 0.15%, and then zinc sulfate .7H O was added for 14 p.p.m. in one section and was not added for another section of the said saccharine materials respectively. After sterilization, 0.6 lit. of flask cultured liquor of Candida utilis was implanted to respective media of 120 lit. each, and culture was carried out under the condition of agitation at 222 rpm. and aeration at 100 lit/min. (Kd=43.5 lpH of the media was controlled with ammonia and sulfuric acid to pH 4.8 as the standard.

The principal effects obtained with respective raw materials were as shown in the following tables:

Yield of yeast RNA Growth Zinc SulfateJlLO (p.p.m.) relative to Content rate consumed sugar (percent) (percent) From this extract, yeast was separated, from which RNA was collected by extraction in the same manner as Example 1.

Various changes may be made to the methods herein described, without departing from the spirit of the invention or the scope of the following claim.

I claim:

A process for producing yeast ribonucleic acid which comprises culturing a yeast in an aqueous nutrient medium under aerobic conditions wherein the oxygen absorption coefficient is at least 10 10 the aqueous nutrient medium being artifically supplemented by Zinc ion at a concentration of at least 0.25 ppm. and having a phosphate ion concentration of at least 0.15% by weight, and extracting and recovering ribonuclei acid from the yeast.

Zinc Time Required Max. Yeast RNA Residual Sulfate to Reach Max. Concentra- Content Sugar 711 0 Yeast Concention (percent) (percent) (percent) (p .p .m.) tration (11L) Molasses of Siamese Origin- 0 9. 5 1. 42 9. 2 0.21 11 10. 0 1. 48 11. 6 0. 22

Molasses of Origin of Philippines- 0 10.0 1. 52 8. 9 0. 18 14 10. 0 l. 51 11.5 0. 18

Saccharified Liquor of Wood 0 13. 0 1. 32 7. 6 0. 31 14 13. 5 1. 29 10. 9 0. 33

From these culture liquor, the mycelia were separated References Cited y the Examine! in the identical manner to Example 1, from which RNA UNITED STATES PATENTS was collected b extraction.

y 3,163,638 12/1964 Mlwa et al. 19s 2s Example 3 OTHER REFERENCES In a stainless aeration-agitation tank with 400 lit. capacity, the effects of addition of zinc ion was examined by carrying out continuous culture with the molasses of Indian origin. The composition of the culture liquor supplied was sugar concentration 6.0%, ammonium sulfate Cook, The Chemistry and Biology of Yeast, Academic Press Inc., New York, 1958, pages 183 and 296 to 305.

A. LOUIS MONACELL, Primary Examiner.

ALVIN E. TANENHOLTZ, Assistant Examiner. 

